New Frontiers in Translational Research in Neuro-Oncology and the Blood-Brain Barrier: Report of the Tenth Annual Blood-Brain Barrier Disruption Consortium Meeting

Vol. 11, 421–428, January 15, 2005 Clinical Cancer Research 421

New Frontiers in Translational Research in Neuro-oncology and the Blood-Brain Barrier: Report of the Tenth Annual Blood-Brain Barrier Disruption Consortium Meeting

Nancy D. Doolittle,1 Lauren E. Abrey,2 nervous system (CNS) diseases. For this reason, a meeting W. Archie Bleyer,3 Steven Brem,4 partially funded by an NIH R13 grant was convened to Thomas P. Davis,5 Paula Dore-Duffy,6 discuss recent advances and future directions in translational research in neuro-oncology and the BBB. Cell biology and Lester R. Drewes,7 Walter A. Hall,8 9 10 transport across the BBB, delivery of agents to the CNS, John M. Hoffman, Agnieszka Korfel, neuroimaging, angiogenesis, immunotherapy, and gene ther- 11 1 Robert Martuza, Leslie L. Muldoon, apy, as well as glioma, primary CNS lymphoma, and David Peereboom,12 Darryl R. Peterson,14 metastases to the CNS were discussed. Transport across the Samuel D. Rabkin,15 Quentin Smith,16 BBB relates to the neurovascular unit, which consists not only Glen H.J. Stevens,13 and Edward A. Neuwelt1 of endothelial cells but also of pericyte, glia, and neuronal elements. 1Department of Neurology, Oregon Health & Science University, Portland, Oregon; 2Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, New York; 3Divisions of Pediatrics and Community Oncology, M.D. Anderson Cancer Center, Houston, Texas; INTRODUCTION 4Department of Neuro-oncology and Neurosurgery, H. Lee Moffitt Cancer Center, Tampa, Florida; 5Department of Pharmacology, Program Although recent basic science advances in understanding in Neurosciences and Physiological Sciences, University of Arizona mechanisms of the blood-brain barrier (BBB) have been Medical School, Tucson, Arizona; 6Department of Neurology, Wayne 7 substantial, much research remains to be done on translating State University, Detroit, Michigan; Department of Biochemistry and cellular mechanisms to improve the treatment of malignant brain Molecular Biology, University of Minnesota School of Medicine, Duluth, Minnesota; 8Department of Neurosurgery, University of tumors and other neurologic diseases. A meeting was convened Minnesota, Minneapolis, Minnesota; 9National Cancer Institute, Cancer on March 17 to 20, 2004 in Sunriver, Oregon, to discuss recent Imaging Program, Bethesda, Maryland; 10Department of Hematology, preclinical and clinical research in neuro-oncology and the BBB. Oncology, and Transfusion Medicine, Charite Campus Benjamin More than 120 basic science and clinical researchers from 50 Franklin, Berlin, Germany; 11Department of Neurosurgery, Massachusetts General Hospital Neurosurgical Service, Harvard Medical institutions attended the meeting. Seventy-five attendees pre- School, Boston, Massachusetts; 12Department of Hematology and sented their research findings. Senior scientists provided over- Medical Oncology and 13Adult Neuro-oncology Brain Tumor Institute, views and led discussions on advances and future directions in 14 Cleveland Clinic Foundation, Cleveland, Ohio; Department of specific neuro-oncology and BBB topics, then summarized the Physiology and Biophysics, Chicago Medical School, North Chicago, discussions for this meeting report. The cell biology of central Illinois; 15Department of Neurosurgery, Massachusetts General Hospital-East Molecular Neurosurgery Laboratory, Harvard Medical nervous system (CNS) endothelial tight junctions is shown in School, Charlestown, Massachusetts; and 16Department of Fig.1; clinical implications of the BBB are the topics of recent Pharmaceutical Sciences, Texas Tech, Amarillo, Texas reviews (1, 2).

BLOOD-BRAIN BARRIER: CELL BIOLOGY AND ABSTRACT TRANSPORT The blood-brain barrier (BBB) presents a major obstacle Recent cellular studies of the BBB have focused on (a) to the treatment of malignant brain tumors and other central molecular properties of transport systems in both luminal and abluminal plasma membranes, (b) new and refined in vitro models, (c) regulation of tight junctions, (d)cellularmechanismsofdisease, and (e) methodologies for global analyses of BBB function. Received 6/17/04; revised 8/18/04; accepted 8/24/04. It was discussed that a molecular approach to defining Grant support: NIH grant 4R13 CA 86959-04 through the National Cancer Institute, the National Institute of Neurological Disorders and transport properties of the BBB has improved our understanding Stroke, and the National Institute of Deafness and Other Communication of organic nutrient delivery to the brain, fluid and electrolyte Disorders. homeostasis, the transcytotic process, and the role of efflux Note: Dr. E. Neuwelt and the Oregon Health & Science University have transporters (1, 2). Net fluid influx and brain extracellular fluid significant financial interests in Adherex, a company that has a homeostasis may be regulated by hormones produced in the commercial interest in the results of this research and technology. This potential conflict of interest has been reviewed and managed by the CNS that influence blood-brain transport. Transcytosis of insulin Oregon Health & Science University Conflict of Interest in Research and transferrin has been well defined, and these pathways have Committee. been utilized for targeted delivery to the brain and brain tumors. Requests for reprints: Edward A. Neuwelt, Department of Neurology, The presence of active efflux transporters in the BBB prevents Oregon Health & Science University, 3181 Southwest Sam Jackson Park Road-L603. Portland, OR 97201. Phone: 503-494-5626; Fax: 1-503 494- many systemically administered drugs from entering the brain 5627; E-mail:[email protected]. and is a major obstacle in designing drugs to treat neurologic D2005 American Association for Cancer Research. disorders (3). In addition, efforts have been directed at developing

Fig. 1 Molecular model of BBB structures. The major proteins of the BBB tight junctions are the transmembrane claudins and occludins, which dimerize with opposite members on adjacent endothelial cells. Zonula occludens protein ZO1 binds both claudins and occludins as well as the less well characterized junctional adhesion molecule. Additional structural support for the tight junction is provided by zonula occludens proteins ZO2 and ZO3, the cingulin linking protein, and the cytoskeletal protein actin. Adherens junctions consist of transmembrane cadherin proteins linked to a, h, and g catenins (cat). Structural support for adherens junctions is provided by a-actinin (act) and vinculin (vin) linking to actin. AF6 and 7H6 are proteins of unknown function associated with ZO1. Reprinted with permission from Elsevier.1

better in vitro models to simulate BBB function in a highly BLOOD-BRAIN BARRIER IN DISEASE controlled environment. Although a compromised BBB has been reported under The relationship between the cytoskeleton and tight junctions numerous pathologic conditions, the role of a disrupted BBB in apparently allows for regulated alterations in BBB function by the pathogenesis of neurologic illnesses is mostly unknown. modifying paracellular transport (1). Recent data indicate that Alteration of the barrier tight junctions is a hallmark of many abnormal permeability changes accompanying ischemia and CNS pathologies, including tumor, stroke, HIV, encephalitis, reperfusion may utilize calcium-dependent processes that alter Alzheimer’s disease, multiple sclerosis, vasogenic edema, and cytoskeletal and tight junctional functions. More importantly, a bacterial meningitis (2). cellular and molecular approach to studying the brain endothelium Mechanisms for BBB breakdown (or tight junction protein has enabled characterization of specific mechanisms for a variety of rearrangement) seem to involve both direct and indirect effect of pathologic states. Diseases may have different initiating mecha- stress responses and inflammatory mediators. Huber et al. (1) and nisms, but a single common pathway to final pathology (2). Witt et al. (5) have used in vitro and in vivo methodologies in Apoptosis seems to play a key role in seemingly divergent concert to examine hypoxia/post-hypoxic reoxygenation to reveal conditions such as ischemia-reperfusion injury (stroke), multiple alterations in the BBB tight junctional protein complex (Fig. 1), sclerosis, and CNS tumors. This implies common pathways resulting in increased paracellular permeability. Transcription including mitochondrial damage and caspase activation. factor alterations have also been noted that indicate significant Meeting participants agreed that a major area to be BBB activation/compensatory mechanisms resulting from addressed will be regulatory mechanisms of transport across hypoxia/post-hypoxic reoxygenation in both in vitro and in vivo the brain endothelial cell. The differences in endothelial cell models. Defining disruption of the BBB under these conditions properties as a function of development, anatomic location, and may provide potential targets for new treatment paradigms. response to disease need to be ascertained. These advances will Data described by Friedman et al. at the meeting suggest have major importance in neuropharmacokinetics and drug that long-lasting BBB opening may result in astrocytic delivery (3). Progress is being made in applying genomics and dysfunction followed by epileptogenesis. Janigro et al. (6) proteomics to BBB function and disease (4). A highly structured described data showing that S100-h is directly related to the experimental approach using proteomics may lead to identifica- extent and temporal sequence of hyperosmotic blood-brain tion of specific cellular mechanisms and therapeutic targets. barrier disruption (BBBD), suggesting that S100-h is a marker of Future studies will likely consider the ‘‘neurovascular unit,’’ BBB function and that elevated S100-h may indicate the defined as the integration of multiple components including the presence of radiologically detectable BBB leakage. Skold et al. endothelial cell, pericyte, basement membrane, and surrounding described that traumatic brain injury leads to an up-regulation of glial and neuronal elements. The neurovascular unit is dependent cell vascular endothelial growth factor (VEGF), VEGF receptor on specific expressed genes and gene products. 1 (VEGFR1) and VEGFR2 mRNA and protein in and around the

lesion. This finding provides important knowledge about the which are small-molecular-weight agents with reactive sulfur mechanisms of posttraumatic angiogenesis and new potential groups. Several issues seem to be important for chemo- targets for therapeutic interventions. protection. There was consensus that knowledge of preclinical BBB (a) Dose of chemoprotectant: N-Acetylcysteine (NAC) models should be applied to human diseases. Changes in requires 400 to 1200 mg/kg, yielding 2 to 15 mmol/L peak neurovascular status and in regional BBB permeability and blood concentration, for chemoprotection in various animal transport systems should be incorporated in clinical thinking models (14, 15). Clinically, sodium thiosulfate is given at 20 to better understand neurologic disease pathophysiology and g/m2 for otoprotection (16). disease-specific differences in BBB cellular mechanisms. (b) Route of administration: An aortic infusion method enhances bone marrow chemoprotection with thiols (15, 17), DELIVERY OF THERAPEUTICS TO THE whereas i.v. or intra-arterial delivery may target chemo- CENTRAL NERVOUS SYSTEM protection to other organs or brain (14). Several strategies were discussed at the meeting for (c) Timing: Sodium thiosulfate is otoprotective when given 4 improving drug delivery to brain tumors through circumven- hours after carboplatin in patients (16) and when given up to tion of the BBB (7, 8). There was agreement that one 8 hours after cisplatin in rats. NAC is bone-marrow protective approach that offers considerable promise is selective only if given before alkylating chemotherapy but shows inhibition of multidrug-resistance transporters, such as otoprotection when given 4 hours after cisplatin (14). P-glycoprotein, multidrug resistance–associated protein, and/ (d) Isomer: For chiral thiols, such as D- versus L-methionine, or breast cancer resistance protein. P-glycoprotein and breast or L- versus D-NAC, the isomer may be important for cellular cancer resistance protein are highly expressed at the BBB and uptake and/or metabolism that affects chemoprotection. act to markedly restrict brain and brain tumor uptake of a (e) Genetic factors: No high-frequency alleles with signif- broad range of anticancer drugs (9). Inhibition of active efflux icant effect conferring susceptibility to chemotherapy- transport has been shown to increase both CNS anticancer induced hearing loss have been detected; however, genes in drug delivery and therapeutic efficacy. For example, Fellner glutathione and antioxidant pathways may have an effect. et al. (10) showed a 90% reduction in tumor volume in nude mice implanted with intracerebral human glioblastoma when A major issue is the potential to interfere with antitumor treated with paclitaxel together with the second-generation efficacy. A rat brain tumor study showed no change in P-glycoprotein pump inhibitor, valspodar (PSC 833). In the chemotherapeutic efficacy when NAC was given before absence of valspodar, paclitaxel was without effect on brain chemotherapy and/or when sodium thiosulfate was given 4 tumor volume. Similarly Kemper et al. (11) reported a 5-fold and 8 hours after chemotherapy (15). Amifostine (WR-2721) elevation in brain paclitaxel uptake in mice treated with the has the advantage of selectively targeting nontumor cells, thus third-generation P-glycoprotein inhibitor elacridar (GF avoiding chemoprotection of cancer, including CNS malignan- 120918). However, efflux transport inhibition can compromise cies (18). However, results from a recently closed Children’s chemotherapeutic drug clearance from plasma, resulting in Oncology Group protocol indicate amifostine lacks otoprotec- elevated plasma anticancer drug concentration and increased tive activity. This may have been because very little amifostine anticancer drug toxicity. Furthermore, efflux transporters have is able to penetrate the intact BBB and the blood-inner ear critical roles in protecting the brain from a wide range of barrier. potentially toxic molecules (3). Thus, patients on efflux There was consensus that new clinical trials should be transporter blockade therapy will need to be closely developed to address the potential for chemoprotection in monitored. Several third-generation multidrug resistance rever- pediatric patients with cancer. Chemoprotection of normal brain sal agents have been developed (e.g., biricodar, elacridar, and may reduce adverse neurotoxic effects of chemotherapy or zosuquidar) that are reported to have less effect on anticancer ionizing radiation, especially in children in whom protection of drug pharmacokinetics than the earlier agents (12). the vulnerable developing CNS is a greater challenge. A phase I Convection-enhanced delivery, by increasing convective trial was proposed for protection of normal brain from cranial fluid flow within the brain interstitial space, distributes the drug radiotherapy with the use of intrathecal amifostine. In a proposed over a greater volume than would be achievable by simple phase III Children’s Oncology Group trial, delayed sodium diffusion. Furthermore, it provides a means of delivering protein thiosulfate will be evaluated in pediatric histologies treated with immunotoxin and nucleic acid therapeutics to the brain. cisplatin chemotherapy. This trial will determine whether However, the approach is invasive, cannot deliver drug over otoprotection can be achieved in the pediatric population without the entire brain, and may result in variable drug delivery in brain reducing antitumor efficacy. regions in which the BBB is compromised, or in tumors (13). ANGIOGENESIS AND ANGIOLYSIS CHEMOPROTECTION AND PEDIATRIC Angiogenesis is important to the survival of many tumors TUMORS including gliomas (19). Antiangiogenesis therapy can theoreti- Chemoprotectants may have a role in increasing chemo- cally overcome two problems in neuro-oncology: drug delivery therapy dose intensity. Preclinical and clinical studies of hearing and drug resistance (20). Normal nonmalignant endothelial cells and bone marrow chemoprotection have been done with thiols, are presumed to be genomically stable, whereas actively

proliferating angiogenic vessels are associated with the multi- therapy. The small molecular weight inhibitor OSI774 is drug resistance (MDR1) phenotype (21). currently being evaluated in a phase II trial with recurrent Brain tumor angiogenesis has traditionally involved glioblastoma multiforme patients and has shown some promise; the tumor cell and the endothelial cell. Dore-Duffy showed however, mechanisms of failure need further evaluation. This the importance of the CNS microvascular pericyte in regulation class of drugs is also being evaluated as a radiosensitizer in of angiogenesis (22). Using a three-dimensional culture combination therapy with temozolomide and radiation. The system, they showed that primary endothelial cells are unable protein-based agent herstatin, which blocks receptor dimeriza- to form new vessels in the absence of pericytes; however, the tion and activation of epidermal growth factor receptors, has addition of pericytes to endothelial cell cultures resulted in been evaluated in a rat glioblastoma model. Herstatin blocked three-dimensional verified vessels. Furthermore, unlike normal growth of intracerebral glioma expressing the normal epidermal pericytes, pericytes in microvessels of glioblastoma express growth factor receptor, whereas in vivo and in vitro growth of VEGF mRNA and constitutively express VEGF protein. Under cells expressing a mutant constitutively active epidermal growth normal conditions, translation of pericyte VEGF protein is factor receptor were resistant to herstatin (27). under tight regulation, whereas in the tumor-altered pericyte, Recent advances in molecular analysis of gliomas have VEGF gene expression may perpetuate angiogenesis in begun to affect treatment strategies (28). For oligodendrogliomas malignant brain tumors. that are 1p19q deleted, the emphasis has moved to treatment Endothelial progenitor cells derived from bone marrow can with temozolomide up front and using radiation as salvage migrate to brain tumor and control angiogenesis (23). At the therapy. meeting, Glod showed that a subset of CD14+ cells could form From a glioma modeling perspective, standardization of an a functional barrier characteristic of the cerebral endothelium. animal model would be beneficial. Fortin et al. discussed a tumor These endothelial-like monocytic cells incorporate themselves implant model using F98 astrocytic cell lines syngeneic to into the neovascular network in xenografts of brain tumors. Nag Fischer rats, which showed predictable and reproducible tumor and colleagues reported on angiopoietin-1 and -2 regulation of growth. There was consensus that more adult patients with vascular homeostasis (24). The angiopoietins function as glioma should be placed on clinical trials whenever possible to ligands for endothelial cell-receptor–specific tyrosine kinase match the enrollment progress in pediatric tumor clinical trials. Tie-2. As a key mediator of breakdown of the BBB, angiopoietin-1 maintains vascular hemostasis and is antiapop- totic, whereas angiopoietin-2 promotes apoptosis after traumatic IMMUNOTHERAPY AND THE BLOOD-BRAIN brain injury. BARRIER Brem presented two novel pharmacologic inhibitors of Malignant gliomas have been difficult to treat with angiogenesis, a-amyloid and GFA-116 (25). GFA-116, a low immunotherapy because of their immunosuppressive nature molecular weight inhibitor of angiogenesis, blocks experimental and the immune privilege provided by the BBB. Immunotherapy growth and metastatic spread of experimental tumors and will be for malignant gliomas includes targeted toxin therapy and evaluated for the treatment of brain tumors. anticancer vaccines (29). Targeted toxins have been primarily Angiolysis is a new approach to therapy that exploits the administered against malignant gliomas because of their ability structural differences between tumor vasculature and normal brain to be administered directly into target tissue by convection- vasculature. Antagonists against cell adhesion molecules, such as enhanced delivery, thereby bypassing the BBB. These agents are VE-cadherin, which mediates endothelial cell interactions, and N- well developed and are currently under investigation in phase III cadherin, which mediates endothelial cell-pericyte interactions, clinical trials that target either the interleukin-13 or transferrin may cause rupture of tumor blood vessels. Studies are in progress receptor. Despite the mature nature of these trials, there are still using serial magnetic resonance imaging (MRI) with high and low concerns regarding basic issues such as the optimal number of molecular weight contrast agents (ferumoxtran-10 versus gado- catheters for infusion and location for catheter placement. It is linium) to evaluate the effect of the angiolytic agent Exherin also unclear whether slow flow (1 AL/h) or fast flow (10 AL/h) (Adherex Technologies Inc, Durham, NC) on the growth and better influence the distance of distribution of drugs such as permeability of rat brain tumor models. doxorubicin or a transferrin-doxorubicin conjugate. Anticancer Future directions include improved methods of imaging vaccines have been developed for a variety of cancers and have angiogenesis and surrogate markers to determine optimal dosage shown both safety and efficacy in early-phase clinical trials and efficacy of antiangiogenesis drugs in clinical trials. Angio- including those against malignant gliomas. In malignant genesis inhibitors may be most effective in combination with gliomas, initial vaccine trials have been dendritic cell–based chemotherapy (21) or with other angiogenesis inhibitors (26). or have used genetic engineering to modify cytotoxic These inhibitors normalize the tumor’s vascular supply, making T-lymphocytes (CTLs) that recognize the interleukin-13 a2 cells less hypoxic, decreasing expression of MDR1,and receptor expressed on malignant glioma cells (30). potentially making brain tumors more susceptible to drug and Transmigration of virally infected monocytes and T cells radiation therapy. across the BBB plays a major role in HIV infection of the brain. Enhanced matrix metalloproteinase (MMP) expression is seen GLIOMA with HIV infection and the statins may reduce the migration of Translational work in gliomas has led to promising new cell-associated virus into the brain by suppressing MMP targeted therapies such as the epidermal growth factor expression. In animals infected with the simian immunodefi- antagonists and their use as sole agents or as part of combination ciency virus, the identification of macrophages or T cells that

produce specific cytokines such as interleukin-6, tumor necrosis offer the possibility of improving our understanding of the factor a, and IFN-g may help elucidate the nature of the immune molecular phenotype of PCNSL. system in the brain. The optimal treatment of PCNSL, particularly tumor localized in the eyes or cerebrospinal fluid space, remains GENE THERAPY controversial. There was consensus that the use of high-dose methotrexate–based chemotherapy with or without the addition Initial gene therapy approaches for brain tumor therapy of whole-brain radiotherapy (WBRT) is the most effective involved therapeutic, gene-expressing, replication-incompetent approach. However, disease control and overall survival are viral vectors; this approach has proven inefficient. Mechanisms inferior to that seen in similarly aggressive extranodal non- of tumor selectivity have included the use of (a) viral gene Hodgkin’s lymphoma suggesting that either the ability to deletion mutants targeting cancer-related pathways, (b) tumor/ administer therapeutic doses is limited by the blood-brain and tissue-specific promoters to control viral replication, and (c) viral blood-cerebrospinal fluid barrier or that there is an inherent coat modifications to engineer tumor-selective uptake. The biological resistance. Furthermore, PCNSL treatment carries a results of early clinical trials with G207 and the challenges for significant risk of neurotoxicity that is generally attributed to the future study were discussed by Martuza (31). Unlike for other combination of high-dose methotrexate and WBRT; however, cancer therapeutics, the BBB or even the blood-tumor barrier some investigators have reported similar rates of neurotoxicity in significantly limits delivery and efficacy. Rabkin et al. studied patients treated with chemotherapy alone (35). intracarotid injection of oncolytic herpes simplex virus vectors after osmotic BBBD for the treatment of metastatic tumors in the Many groups have sought to improve outcome by intensi- brain in mouse models. They showed that BBBD is necessary for fying the delivery and dose of chemotherapy administered. efficient delivery of herpes vectors into brain tumors. Muldoon BBBD, high-dose chemotherapy with autologous stem cell et al. have shown that the distribution of dextran-coated iron support, and use of monthly maintenance chemotherapy are all oxide particles the size of the adenoassociated virus in rat brain strategies that have been used with varying but comparable mimics adenovirus and herpes virus distribution, allowing MRI degrees of success (36–39) and are evaluated in ongoing trials. of virus delivery (32). BBBD-enhanced methotrexate delivery has shown increased All gene therapy strategies depend on having defined targets. CNS dose intensity (40), with 86% of patients in complete Stanimirovic et al. showed that unique ‘‘targets’’ including response at 1 year demonstrating no cognitive loss (39). New vascular targets for tumor-selective drug and gene therapy could Institutional Review Board–approved protocols are under way be identified by molecular fingerprinting of glioblastoma vessels using BBBD to (a) deliver rituximab (an anti-CD20 monoclonal using a combination of laser capture microdissection, genomics, antibody) across the BBB in recurrent PCNSL (Fig.2) and (b) and proteomics (33). Mukhtar et al. described a new gene therapy clear CNS relapse before systemic high-dose chemotherapy with vector for the brain based on spleen necrosis virus, which can peripheral blood stem cell rescue in patients with systemic non- integrate into nondividing cells. Pseudotyping with rabies virus Hodgkin’s lymphoma. In an effort to eradicate disease involving glycoproteins allowed targeting to neural cells. the vitreal or cerebrospinal fluid compartment, investigators are Jensen described a novel genetic engineering approach for exploring the delivery of chemotherapy directly into the eye redirecting the antigen specificity of CD8+ cytolytic T cells to the (41) as well as the delivery of rituximab into the cerebrospinal glioma-restricted interleukin-13 a2 cytokine receptor and the fluid (42). application of these technologies to a clinical adoptive therapy An international effort to improve the understanding and trial using autologous IL13Ra2-specific CTL clones. Genetic treatment of PCNSL has resulted in the establishment of the engineering was used to express varied luciferases in both tumor International PCNSL Collaborative Group (43). Efforts are under cells and lymphocytes to track cell migration using optical way to establish tissue databanks for molecular analysis, to imaging. standardize clinical trial reporting and to establish a prospective As discussed at the meeting, a broad range of gene therapy patient database. approaches is being pursued. Their success will depend on further identification of molecular targets, efficient delivery to CENTRAL NERVOUS SYSTEM METASTASES the brain, and imaging of the vector, gene expression, and There was consensus that CNS metastases is an increasing disease end points. clinical problem. The discussion on brain metastases emphasized (a) landmark and future therapeutic trials, (b) regional perfusion PRIMARY CENTRAL NERVOUS SYSTEM studies, and (c) the biology of brain metastases. For certain LYMPHOMA subgroups of patients, radiation sensitizers such as motexafin- Non-AIDs primary CNS lymphoma (PCNSL) is a rare type gadolinium and efaproxiral with WBRT is superior to WBRT of extranodal non-Hodgkin’s lymphoma that occurs exclusively alone (44). Several randomized trials of chemotherapy alone or in the brain, eyes, or spinal fluid. Although more than 90% of with WBRT have shown higher response rates but no survival PCNSL is of B- cell origin, the molecular characteristics are advantage. Current studies will compare WBRT plus temozolo- poorly understood, in stark contrast to systemic non-Hodgkin’s mide or topotecan, respectively, with WBRT alone. lymphoma. Preliminary evidence suggests that PCNSL shares It was agreed that further trials of chemotherapy, particu- some characteristics of the germinal center phenotype and that larly preirradiation, should be pursued. New phase I and II the expression of bcl-6 may have prognostic importance (34). studies utilize intra-arterial chemotherapy, dose-intense temozo- New techniques such as comparative genomic hybridization lomide + vinorelbine (recurrent), temozolomide + thalidomide +

Fig. 2 MRI of a subject with recurrent non-AIDs PCNSL (arrows), who previously failed methotrexate-based chemotherapy. A and B, axial T1 with gadolinium enhancement and coronal T1 views (January 27, 2004) before rituximab in conjunction with carboplatin-based BBBD treatment. C and D, the same sequences and views approximately 4 weeks after treatment with rituximab (February 25, 2004) demonstrating a complete response.

1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (melanoma) or treatment of micrometastatic disease in the brain, a goal that will intrathecal radioactive ligands (leptomeningeal carcinoma). require delivery across an intact BBB. Newton et al. (45) described results using intra-arterial carboplatin and i.v. etoposide in patients who had prior irradiation for a NEUROIMAGING variety of brain metastases. Thirteen of 24 evaluable patients As neuroimaging techniques have developed and evolved had objective responses (54.2%) with a median time to they have become increasingly important in assessing the progression of 30 weeks in responders (range, 6-118 weeks), biological and physiologic properties of brain tumors. Imaging and the treatment was well tolerated (45). A study is under studies now allow for determination of important clinical development for patients with metastatic breast cancer to the parameters such as perfusion, metabolism, proliferative activity, CNS, utilizing BBBD to deliver herceptin across the BBB in and vascular permeability. Neuroimaging assessments, although HER-2/neu positive patients. Participants agreed that quality of important, have not provided information that has significantly survival is paramount; therefore, clinical trials in CNS improved brain tumor survival. The following advances are metastases must include quality of life as well as neuro- needed to affect survival: (a) the ability to image infiltrative psychometric end points. disease, (b) better assessment of the true extent of disease and Studies of the regional behavior of brain metastases using the actual tumor volume, and (c)molecularbiological dynamic perfusion computed tomography scans have correlated information relevant to treatment strategies. symptoms with altered regional perfusion during WBRT. Future An overview of the current state of brain tumor imaging studies will use dynamic perfusion MRI to better define the local included (a) recent data on fluorodeoxyglucose positron emission behavior of brain metastases. Elucidation of the metastatic tomography (PET) and its prognostic capabilities, (b) new PET cascade as well as the genetic control of metastasis should lead tracers and how they may be helpful in differentiating low-grade to the development of rational and directed therapies. For from high-grade tumors as well as differentiating radiation example, discovery of the role of MMP in tumor cell invasion necrosis from recurrent tumor (46), (c) new MRI contrast agents after tumor cells breach the BBB suggests a strategy using MMP that show superior image quality (47), and (d) a recent publication inhibitors to inhibit tumor cell invasion. Such a strategy could be showing the utility of an optical imaging technique that could used as treatment, as an adjunct to initial definitive therapy, or as improve brain tumor delineation (48). Two presentations focused an adjunct to WBRT for established brain metastases. An on high-field-strength MRI (8 T), which allowed for high- important goal of future trials should be primary prevention or resolution imaging of tumor microvasculature and delineation of

Fig. 3 Saggital (A) and transaxial (B) MRI views of two i.v. contrast agents (gadolinium and ferumoxtran-10), which do not cross the normal BBB, using coregistration by Brain Voyager QX (Brain Innovations, Maastricht, Netherlands) in a subject with PCNSL. Two different image sets are reconstructed. The enhancement of the two contrast agents is color coded, and the two image sets are aligned and added to each other. Yellow areas, enhancement with both gadolinium and ferumoxtran-10; green areas, enhancement only with ferumoxtran- 10. The volume of enhancement of tumor is greater with ferumoxtran-10, a dextran- coated iron oxide crystal, which is superparamagnetic.

areas of increased cellularity and vascular proliferation. It was felt Thomas Jacobs, Damir Janigro, Michael Jensen, Georgette Kanmogne, that this information could be used preoperatively in selecting Kristin Knight, Agnieszka Korfel, Dale Kraemer, Steven Krosnick, sites to perform image-guided stereotactic biopsy. Andrew Maclean, Christopher Madden, Sandor Manninger, In an animal imaging experiment at 8 T, it was shown that by Lorraine Marin, Robert Martuza, David Mathieu, John McGregor, using an ultrasmall iron oxide particle (USPIO) contrast agent Emmanuelle Meuillet, Barbara-Ann Millar, David Miller, Robert Miller, Maria Moreno, Pierre Mourad, Muhammad Mukhtar, Leslie Muldoon, there was additional improvement in imaging tumor microvascu- Tulio Murillo, Sukriti Nag, H. Stacy Nicholson, Edward Neuwelt, lature. In a separate presentation using another USPIO contrast Herbert Newton, Brian O’Neill, Peter Orbay, Michael Pagel, Weihong Pan, agent in patients with brain tumor (49), investigators showed David Peereboom, Darryl Peterson, Scott Plotkin, Brad Pollock, larger areas of enhancement in some cases (Fig. 3). With delayed Johnny Pryor, Amina Qutub, Samuel Rabkin, William Rooney, imaging, the USPIO is taken up into tumor macrophages and Robert Rostomily, James Rubenstein, Nathan Selden, Robert Shangraw, reactive astrocytes, as visualized histochemically with iron Ian Simpson, Mattias Skold, Matthew Slater, Justine Smith, Quentin Smith, staining (50). Due to the virus-like size of the USPIO it may be Carole Soussain, Danica Stanimirovic, Glen Stevens, Edward Stopa, Lillian Sung, Michael Toborek, Rose Marie Tyson, Michael Vogelbaum, possible to monitor penetration of viral vectors when using gene Katherine Wild, Kathryn Woodbury-Harris, Jeffrey Wu, Byron Young. therapy. It was agreed that newer molecular imaging techniques should be integrated into brain tumor management, thus providing REFERENCES critical information that may significantly improve the survival and care of patients with brain tumors. 1. Huber JD, Egleton RD, Davis TP. Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier. Trends Neurosci 2001;24:719–25. CONCLUSION 2. Neuwelt EA. Mechanisms of disease: the blood-brain barrier. With regard to CNS malignancy, endothelial cells along with Neurosurgery 2004;54:131–42. glia, pericytes, and neurons should be viewed as a neurovascular unit 3. Taylor EM. The impact of efflux transporters in the brain on the development of drugs for CNS disorders. Clin Pharmacokinet that impedes delivery of therapeutics. Delivery of most chemother- 2002;41:81–92. apy across the BBB is modest, and macromolecule and gene vector 4. Shusta EV,Boado RJ, Mathern GW, Pardridge WM. Vasculargenomics delivery to the CNS are an order of magnitude more difficult than of the human brain. J Cereb Blood Flow Metab 2002;22: 245–52. drug delivery. Translational research involving the neurovascular 5. Witt KA, Mark KS, Hom S, Davis TP. Effects of hypoxia- unit is greatly needed because the CNS is, to a large extent, a reoxygenation on rat blood-brain barrier permeability and tight junctional sanctuary, ‘‘immune’’ from many neuro-oncologic therapies. protein expression. Am J Physiol Heart Circ Physiol 2003;285: H2820–31. ACKNOWLEDGMENTS 6. Marchi N, Cavaglia M, Fazio V, Bhudia S, Hallene K, Janigro D. Peripheral markers of blood-brain barrier damage. Clin Chim Acta Data presented at the meeting may or may not have been previously 2004;342:1–12. published. We thank Kristina Stromvig, Lisa Bennett, Nan Hedrick, and 7. Lee HJ, Pardridge WM. Monoclonal antibody radiopharmaceuticals: Debbie Bird for coordinating the meeting and the following for their cationization, pegylation, radiometal chelation, pharmacokinetics, and research presentations and for facilitating discussion: Lauren Abrey, tumor imaging. Bioconjug Chem 2003;14:546–53. Clarke Anderson, Richard Aplenc, Tracy Batchelor, Gretchen Belenchia, 8. Ningaraj NS, Rao MK, Black KL. Adenosine 5V-triphosphate- Susan Bell, W. Archie Bleyer, William Bodell, Dennis Bourdette, sensitive potassium channel-mediated blood-brain barrier permeability Rita Braziel, Steven Brem, Joseph Bubalo, Bob Carter, Thomas Chen, increase in a rat brain tumor model. Cancer Res 2003;63:8899–911. Gregory Christoforidis, Gail Clinton, Thomas Davis, Tom Dickey, 9. Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, Gottesman MM. P- Amy Donahue, Nancy Doolittle, Paula Dore-Duffy, Lester Drewes, glycoprotein: from genomics to mechanism. Oncogene 2003;22:7468–85. David Fortin, Gert Fricker, Alon Friedman, Barbara Glidewell, John Glod, 10. Fellner S, Bauer B, Miller DS, et al. Transport of paclitaxel (Taxol) Gerry Grant, Michael Guarnieri, Mary Kay Gumerlock, Walter Hall, across the blood-brain barrier in vitro and in vivo. J Clin Invest John Hoffman, Gregory Hornig, Matthew Hunt, Paula Jacobs, 2002;110:1309–18.

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